Use: It should be possible to selectively block degradation of PKC without affecting the membrane associated activation. This will allow an assessment of the role of proteolysis in the activation of the protein kinase pathway.

Applications

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application

Abreviews

Notes

ELISA

Use at an assay dependent concentration.

ICC

Use at an assay dependent concentration.

IP

Use at an assay dependent concentration.

RIA

Use at an assay dependent concentration.

WB

Use at an assay dependent concentration.

Flow Cyt

Use 1µg for 106 cells.

IHC-FoFr

Use at an assay dependent concentration. PubMed: 18368118

IHC-Fr

1/500.

IHC-P

Use at an assay dependent concentration. PubMed: 17266784

Target

RelevanceProtein Kinase C (PKC) isoforms are serine/threonine kinases involved in signal transduction pathways that govern a wide range of physiological processes including differentiation, proliferation, gene expression, brain function, membrane transport and the organization of cytoskeletal and extracellular matrix proteins. Increasing evidence from studies using in vitro and in vivo systems points to PKC as a key regulator of critical cell cycle transitions, including cell cycle entry and exit and the G1 and G2 checkpoints. PKC-mediated control of these transitions can be negative or positive, depending on the timing of PKC activation during the cell cycle and on the specific PKC isozymes involved. There have been at least 12 different PKC isoforms identified in humans to date, including alpha, beta I, beta II, gamma, delta, epsilon, zeta, eta, theta, iota, lambda, and mu.

Anti-PKC antibody [MC5] images

Flow Cytometry - Anti-PKC antibody [MC5] (ab31)

Overlay histogram showing Jurkat cells stained with ab31 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab31, 1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was a goat anti-mouse Alexa Fluor® 488 (IgG; H+L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HL60 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunocytochemistry/ Immunofluorescence - PKC antibody [MC5] (ab31)

Vero cells with ab31 at a 1/50 dilution. This picture was kindly submitted as part of the review by Martha Simpson-Holley.

Immunocytochemistry/ Immunofluorescence - PKC antibody [MC5] (ab31)

293 T cells with ab31 at 1/50 dilution. This picture was kindly submitted as part of the review by Martha Simpson-Holley.

ab31 staining PKC in Mouse spleen and duodenum tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/500 in PBS) for 8 hours at 4°C. An Alexa Fluor®488-conjugated Goat anti-mouse IgG polyclonal (1/1000) was used as the secondary antibody. Nuclei were stained by DAPI.

Indeed, this antibody is a clone and is used by many researchers. However I could not conclude on its specificity. Indeed, some sources quote that this antibody is specific for PKC alpha, whereas other state that there might be crossreactivity with PKC...

Thank you for your enquiry. For ab4127, this antibody was validated on tPA induced myeloid cells for IHC. Also, melanocytes have been known to express PKC alpha and alternatively brain tissue is typically inherently expressive.
For ab31, I regret t...

I apologize for the delay in responding to your enquiry. The originator of this antibody attests that this antibody has been found to recognize Protein kinase Ca. Epitope: within the variable region V3 PKCa (the site of limited proteolysis on the nativ...

We have no information about the dilutions originally used with this antibody. I would suggest trying the antibody at 1:1000, 1:250 and 1:2000 initially, the ideal dilution will depend on the details of your experiments.

Abcam Scientific Support

Answered on Jul 11 2002

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"